N-glycans have been implicated in a large number of biological processes including cell adhesion. However, the exact role of N-glycans in vivo is largely undefined. In this regard, genetic diseases caused by glycosylation defects will serve as useful models for elucidating the role of N-glycans. To date, two human genetic diseases in this category are known. They are carbohydrate deficiency glycoprotein syndrome and HEMPAS (hereditary erythroblastic multinuclearity with positive acidified serum lysis). HEMPAS is a rare genetic disease caused by a defective Golgi alpha-mannosidase II, leading to incomplete glycosylation of polylactosaminoglycans in erythroid cells. HEMPAS patients are anemic and show liver cirrhosis, diabetes and gall stones, suggesting that defective synthesis of N-glycans affects these tissues. To understand more fully the role of complex N-glycans in development and diseases, we propose the following studies. First, we will determine mutations in the gene of alpha-mannosidase II (alphaMII) in two HEMPAS cases. One patient showed significant reduction of alpha-MII mRNA. Another patient expressed normal levels of alpha-MII mRNA, while no alpha- MII enzymatic activity was detected. Mutation of the alpha-MII gene in these cases will be determined. Second, we will define substrate specificity and cell type-specific expression of alpha-MillX, a new alpha- MII isozyme we have recently identified. Expression of alpha-MIIX in various cell types will be examined to explain the tissue specific manifestation of HEMPAS disease. Third, we will create a mouse strain in which the alpha-MIIX gene is knocked-out. This strain and the alpha-MII gene knock-out mouse strain will be used as an animal model of HEMPAS. The glycosylation pattern and pathology of the gene knocked-out mice will be compared with those from HEMPAS patients. Finally, we will evaluate the effect of alpha-MII and/or alpha-MIIX null mutation on carbohydrate mediated cell adhesion. Cells from alpha-Mill and/or alpha-MIIX mice will be examined for their binding to E-, P-, and L-selectins. Creating alpha MII/alpha-MIIX gene knock-out mice will provide materials for functional assessments of N-glycans in ontogeny and post-natal function, with the expectation that to gain a better understanding HEMPAS disease. Moreover, this proposal will explore the involvement of N-glycans in carbohydrate mediated cell adhesions in vivo.